Properties of papain, immobilized on organosilica

Papain, a proteolytic enzyme, is used in the reactions of organic synthesis for preparing peptides. The use of immobilized papain with this aim is very promising. Preparations of papain immobilized by organosilica have been studied for their physicochemical properties as well kinetics of the papain immobilization by amino-organosilica activated by cyanuric chloride. Retention of the enzyme activity of immobilized papain reached 40% and depended on the amount of enzyme bound with the carrier. Kmobs of the immobilized enzyme did not differ significantly from that of the soluble enzyme. After immobilization the pH-optimum an pH-profile of the catalytical activity of papain remained unchangeable. For the period of 20 days immobilized papain has lost 20-50% activity.     

金属螯合载体定向固定化木瓜蛋白酶的研究

以磁性金属螯合琼脂糖微球为载体,利用金属螯合配体（IDACu²⁺）与蛋白质表面供电子氨基酸相互作用的原理,定向固定了木瓜蛋白酶。固定化最适条件为Cu²⁺1.5×10^-2mol/g载体、固定化时间4h、固定化pH7.0、给酶量30mg/g载体。固定化酶的最适反应温度70℃、最适反应pH8.0,固定化酶的热稳定性明显高于溶液酶,固定化酶活力回收为68.4％,且有较好的操作稳定性,载体重复使用5次后固定化酶酶活为首次固定化酶79.71%。     

Hydrolysis of soyabean by papain immobilized on wood by radiation polymerization

Summary Papain was immobilized on wood chips by radiation polymerization without substantial loss of enzyme activity. The immobilized papain was used to hydrolyse soyabean meal and found to be stable upto 6 cycles of operation. Maximum hydrolysis occurred with 15% (W/V) immobilized matrix.     

Immobilized papain on gold nanorods as heterogeneous biocatalysts

Papain, a thiol protease present in the latex of Carica papaya, is an enzyme which exhibits broad proteolytic activity, and, for this reason, it is utilized in a variety of industrial applications. Immobilization of papain on gold nanoparticles highly preserves its activity and enhances the stability, allowing the reuse of the linked enzyme many times without any significant loss of its catalytic performance. In particular, k _cat and K _M values remain substantially unchanged, while immobilized form shows a higher activity on a wider pH range retains 80 % residual activity also at 90 °C and shows higher functionality than the free form when incubated for long time (1 h) at 90 °C and at extreme pH values (3 and 12). A higher activity of immobilized papain with respect to the free form in the presence of various bivalent metal ions, known as strong inhibitors of papain, was also found. The reasons of this enhanced stability of gold nanorods immobilized papain are discussed.     

Chemical modification of papain for use in alkaline medium

Chemical modification is a useful method to recognize and modify functional determinants of enzymes. Papain, an endolytic cysteine protease (EC3.4.22.2) from Carica papaya latex has been chemically modified using different dicarboxylic anhydrides of citraconic, phthalic, maleic and succinic acids. These anhydrides reacted with five to six amino groups of the lysine residues in the enzyme, thereby changing the net charge of the enzyme from positive to negative. The resultant enzyme had its optimum pH shifted from 7 to 9 and change in temperature optima from 60 to 80 °C. The modified papain also had a higher thermostability. Stability of the modified papain was further increased by immobilization of the enzyme either by adsorption onto inert matrix or by entrapment in polysaccharide polymeric gels. Entrapment in starch gel showed better retention of enzyme activity. Incorporation of modified and immobilized enzymes to branded domestic detergent powders was found to have very good activity retention. The papain entrapped in starch gel showed better stability and activity retention than in other carbohydrate polymers when added to domestic detergent powders.     

Chemically modified papain for applications in detergent formulations

Papain was modified using succinic anhydride, and the modified papain so obtained was compared with the native papain for its activity and stability in detergents. This study was done using commercial enzyme detergents as references. It was found that modified papain retained activity comparable to the commercial enzyme detergents. Chemically modified papain may prove to be an inexpensive alternative to alkaline proteases that are used in detergents.     

壳聚糖固定化木瓜蛋白酶的研究

以壳聚糖为载体,成二醛为交联剂将木瓜蛋白酶固定化。5％戊二醛在4-6℃下处理载体5h,加酶液(3.5mg/mL蛋白,pH7.2)固定12h,活力回收达32％,作用于酪蛋白的半衰期为36天,其表观K_m(酪蛋白)值为0.075％(W/V),溶液酶的K_m值为0.086％;最适pH7.0～7.5,溶液酶为7.0～8.5。固定化酶在pH8.5以下,溶液酶在9.0以下活力稳定。固定化酶在45℃以下,溶液酶在75℃以下稳定。用6mol/L脲洗脱固定化酶4次(5.5h)活力仍有54.5％。用固定化酶处理啤酒浊度比对照下降了1.5-3.7倍,蛋白质含量下降了44％,冷藏(4℃)120天无冷混浊现象发生并保持了啤酒原有风味和理化性状。     

Preparation and properties of immobilized papain and lipase.

Papain and lipase were immobilized on derivatized Sepharose 4-B. The activated agarose had a binding capacity of 1.2 micronmol amino groups/ml packed agarose or 17 mg proteins/g dry agarose. The immobilized enzyme preparations were tested for the effects of pH of assay, temperature of assay, and substrate concentrations. The effect of 6M urea on the activity of papain was also determined. Soluble forms of the enzymes were used for comparison. Immobilization of the enzymes resulted in slightly different pH and temperature optima for activities. For immobilized papain Km(app) was similar to the one observed with soluble papain. Immobilization of lipase, however, cause a decrease in Km values. The immobilized enzyme preparations were stable when stored at 4 degrees C and pH 7.5 for periods up to eight months. The soluble enzymes lost their activity within 96 hr under similar storage conditions. Immobilized papain did not lose any activity after treatment with 6M urea for 270 min, whereas soluble papain lost 81% of its activity after the urea treatment, indicating that the immobilization of papain imparted structural and conformational stability to this enzyme.     

Fibrous polymer-grafted chitosan/clay composite beads as a carrier for immobilization of papain and its usability for mercury elimination

Papain, which is an industrially important enzyme, has been immobilized on fibrous polymer-modified composite beads, namely poly(methacrylic acid)-grafted chitosan/clay. Characterization studies have been done using FTIR and SEM analysis. Operating parameters such as pH and initial concentration of papain have been varied to obtain the finest papain immobilized polymer-modified composite beads. The immobilization capacity of composite beads has been determined as 34.47 ± 1.18 (n = 3) mg/g. The proteolytic activity of immobilized papain was operated using bovine serum albumin (BSA) and maximum velocity (V _max) and Michaelis–Menten constant (K_m) values of the free and immobilized enzymes were determined using Lineweaver–Burk and Eadie–Hofstee equations. Usability of papain immobilized polymer-modified composite beads as adsorbents for the elimination of mercury was investigated. The maximum removal capacity of PIPMC beads has been found to be 4.88 ± 0.21 mg Hg/g when the initial metal concentration and weight of polymer-modified composite beads were 50 mg/L and 0.04 g at pH 7, respectively. Mercury removal performance of the papain immobilized polymer-modified composite beads was investigated in conjunction with Cu (II), Zn (II) and Cd (II) ions. The mercury adsorption capacity of papain immobilized polymer-modified composite beads was a slight reduction from 1.15 to 0.89 mg/g in presence of multiple metal salts.     

木瓜蛋白酶在飘珠上的固定化和红外活化

用火电厂的废物——飘珠作载体,将木瓜蛋白酶固定化得到成功,比活力为天然酶的26.6％。但发现,采用适当的红外辐射又可使固定化酶活力提高约30％。     

高分子膜载体固定化木瓜蛋白酶

在浸润条件下,以0.5%(v/v)戊二醛交联的高分子膜尼龙载体固定化木瓜蛋白酶。对固定化条件进行了优化,比较了固定化酶与游离酶的酶学参数。结果表明,4℃、pH6.0条件下,将膜载体浸润于2mg/mL酶液中5h,固定化酶活为303.4U/g。固定化酶最适反应pH为6.0～7.0,最适反应温度为65℃。其pH稳定性、热稳定性均比游离酶高。     

Protein immobilization to alumina supports: II. Papain immobilization to alumina via organophosphate linkers

Particulate aluminum oxides (alumina) were examined as supports for the immobilization of the proteolytic enzyme papain. Two alumina supports termed C1 and CPC were derivatized using organic phosphate linkers to create free carboxyl groups using a two-step process. Papain binding to these derivatized aluminas was performed using the water soluble carbodiimide 1-ethyl-3-(dimethylaminopropyl) carbodiimide. Reactions were optimal at 10 mM carbodiimide. The immobilized protein showed similar kinetic constants when compared to the solution protein. The pH dependence and thermal stability were essentially identical. The immobilized papain showed a blue shift in the intrinsic fluorescence emission maxima. Papain modified with the active site-specific fluorescent probe acrylodan showed overlapping emission maxima. These results are interpreted as retention of the hydrophobic environment of the active site with a perturbation in the structure of the rest of the protein caused by its association with the negatively charged surface. (c) 1992 John Wiley & Sons, Inc.     

Purification and in situ immobilization of papain with aqueous two-phase system

Papain was purified from spray-dried Carica papaya latex using aqueous two-phase system (ATPS). Then it was recovered from PEG phase by in situ immobilization or preparing cross-linked enzyme aggregates (CLEAs). The Plackett-Burman design and the central composite design (CCD) together with the response surface methodology (RSM) were used to optimize the APTS processes. The highly purified papain (96-100%) was achieved under the optimized conditions: 40% (w/w) 15 mg/ml enzyme solution, 14.33-17.65% (w/w) PEG 6000, 14.27-14.42% (w/w) NaH2PO4/K2HPO4 and pH 5.77-6.30 at 20°C. An in situ enzyme immobilization approach, carried out by directly dispersing aminated supports and chitosan beads into the PEG phase, was investigated to recover papain, in which a high immobilization yield (>90%) and activity recovery (>40%) was obtained. Moreover, CLEAs were successfully used in recovering papain from PEG phase with a hydrolytic activity hundreds times higher than the carrier-bound immobilized papain.     

Preparation and characterization of Rhizopus amyloglucosidase immobilized on poly(o-toluidine)

The starch hydrolyzing enzyme amyloglucosidase (AMG) from Rhizopus was immobilized onto the protonated salt (TS) and basic (TB) forms of chemically synthesized poly(o-toluidine) (POT) using adsorption and covalent binding. The polymers were activated with glutaraldehyde prior to covalent bonding. The immobilization efficiency was affected by the pH of the immobilization medium, contact time and amount of enzyme. After immobilization, the pH and temperature were changed to conditions under which the enzyme is most active. Immobilized AMG was more stable with respect to changes in pH and increases in temperature compared to free AMG. The immobilized enzyme retained high catalytic activity after multiple uses and showed enhanced stability with storage compared to free enzyme.     

Improved stability and catalytic activity of chemically modified papain in aqueous organic solvents

Papain was modified with the anhydrides of various monocarboxylic (acetic or propionic) and dicarboxylic (citraconic, maleic or succinic) acids. 7–10 of the 11 primary amino groups of the enzyme were modified. The organic solvent tolerances of the modified enzyme forms were increased (especially in the concentration range of 10–60%) in comparison with the unmodified enzyme. Acylation enhanced the catalytic activity and stability of papain both in buffer and in aqueous organic solvents (ethanol and acetonitrile). Decrease of the positive charges on the surface of papain resulted in a higher enzyme stability than when they were replaced by negative charges. The kinetic parameters revealed that in aqueous ethanol the maximum rates (V_max) and Michaelis constants (K_M) of the modified papain forms were increased, and higher catalytic efficiencies (k_cat/K_M) were detected as compared with the native enzyme. The results of near-UV circular dichroism and tryptophan fluorescence spectroscopic studies suggested that the modifications caused only local changes around the aromatic residues. The modified enzyme forms led to higher N-acetyl-l-tyrosine ethyl ester synthesis conversions in aqueous ethanol; acetyl and propionyl papain furnishing the highest productivity.     

Immobilization of Papain on the Mesoporous Molecular Sieve MCM‐48

The immobilization of papain on the mesoporous molecular sieve MCM‐48 (with a pore size of 6.2 nm in diameter) with the aid of glutaraldehyde, and the characteristics of this immobilized papain are described. The optimum conditions for immobilization were as follows: 20 mg native free enzyme/g of the MCM‐48 and 0.75 % glutaraldehyde, 2 h at 10–20 °C and pH 7.0. Under these optimum conditions for immobilization, the activity yield [%] of the immobilized enzyme was around 70 %. The influence of the pH on the activity of the immobilized enzyme was much lower compared to the free enzyme. The thermostability of the immobilized enzyme, whose half‐life was more than 2500 min, was greatly improved and was found to be significantly higher than that of the free enzyme (about 80 min). The immobilized enzyme also showed good operational stability, and the activity of the immobilized enzyme continued to maintain 76.5 % of the initial activity even after a 12‐day continuous operation. Moreover, the immobilized enzyme still exhibited good storage stability. From these results, papain immobilized on the MCM‐48 with the aid of glutaraldehyde, can be used as a high‐performance biocatalyst in biotechnological processing, in particular in industrial and medical applications.     

Papain,chymotrypsin and related proteins—a comparative study of their beer chill-proofing abilities and characteristics

The role of proteolysis in the beer chill-proofing action of the proteolytic enzyme papain (EC 3.4.22.2) has been investigated by comparing the chill-proofing ability of papain with that of a proteolytically inactive derivative, S-carboxymethylpapain. The latter was prepared by treating papain with bromoacetic acid to carboxymethylate selectively the single essential sulphydryl group of the enzyme, that of l-cysteine-25. Both papain and S-carboxymethylpapain were found to exhibit increasing chill-proofing ability with increasing concentration in beer; at a protein concentration in beer of 30 μg/ml the chill-proofing effect of each protein proved to be substantial. Papain, either in the presence or absence of sodium bisulphite, was, however, found to be more effective than S-carboxymethylpapain at all protein concentrations. It is concluded that the chill-proofing action of papain originates largely, but not wholly, from its proteolytic action. Similarly, the chill-proofing ability of the proteolytic enzyme chymotrypsin (EC 3.4.21.1) has been compared with that of its proteolytically inactive zymogen, chymotrypsinogen A. Both proteins were found to exhibit increasing chill-proofing ability with increasing concentration in beer. The chill-proofing effect of chymotrypsin was, however, found to be greater than that of chymotrypsinogen A at all protein concentrations in beer. On the basis of these results and the close similarities in the molecular structures of chymotrypsin and chymotrypsinogen A, it is concluded that the chill-proofing action of chymotrypsin also originates largely, but not wholly, in its proteolytic action. The results from this study collectively demonstrate that no straightforward correlation exists between the proteolytic activity added to beer and its resistance to chill-haze formation.     

Preparation of immobilized papain covalently bound on natural cellulose for treatment of beer

Summary Papain was covalently immobilized on both natural wood chip and cotton without substantial loss of enzymic activity, and its optimum pH was 6–10; optimum temperature, 67°C. The immobilized papain showed high activity for hydrolysis of beer peptide and was used in treatment of beer for 28 days without loss of activity.     

Preparation and properties of soluble-insoluble immobilized proteases

In order to carry out an effective enzyme reaction, the preparation of soluble-insoluble immobilized enzyme was investigated. Proteases were selected as model enzymes, and their immobilization was carried out by using an enteric coating polymer as a carrier. Among the polymers tested, methacrylic acid-methylacrylate-methylmethacrylate copolymer (MPM-06) gave the most active soluble-insoluble immobilized papain. This immobilized papain showed insoluble from below pH 4.8 and soluble form above pH 5.8; it was also soluble in water-miscible organic solvent. It was reusable and more stable with heat and water-miscible organic solvents than native proteases. Furthermore, various proteases could be immobilized by using MPM-06 with high activity. Chymotrypsin immobilized by this method catalyzed the effective peptide synthesis in a heterogeneous reaction system containing water-miscible organic solvent.     

A novel method for the immobilization of glucoamylase onto polyglutaraldehyde-activated gelatin

A novel method was developed for the immobilization of glucoamylase from Aspergillus niger. The enzyme was immobilized onto polyglutaraldehyde-activated gelatin particles in the presence of polyethylene glycol and soluble gelatin, resulting in 85% immobilization yield. The immobilized enzyme has been fully active for 30 days. In addition, the immobilized enzyme retained 90 and 75% of its activity in 60 and 90 days, respectively. The enzyme optimum conditions were not affected by immobilization and the optimum pH and temperature for free and immobilized enzyme were 4 and 65 °C, respectively. The kinetic parameters for the hydrolysis of maltodextrin by free and immobilized glucoamylase were also determined. The K_m values for free and immobilized enzyme were 7.5 and 10.1 g maltodextrin/l, respectively. The V_max values for free and immobilized enzyme were estimated as 20 and 16 μmol glucose/(min μl enzyme), respectively. The newly developed method is simple yet effective and could be used for the immobilization of some other enzymes.